Orientador: Giselle Zenker Justo / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-18T14:42:19Z (GMT). No. of bitstreams: 1 Silva_RodrigoAugustoda_D.pdf: 6436030 bytes, checksum: 9f8fa7c9b01f00bc5d2d2396d4099a1e (MD5) Previous issue date: 2011 / Resumo: A epiderme é constantemente confrontada por inúmeros agentes estressores. Variações na umidade ou exposição à radiação ultravioleta afetam o balanço osmótico e o estado redox celular alterando, assim, as características fisiológicas da pele. Em resposta aos diferentes estímulos os queratinócitos ativam vias distintas de sinalização. Portanto, o balanço entre as vias de sobrevivência e morte determina o destino celular. A fim de se determinar possíveis alvos moleculares associados a morte e sobrevivência de queratinócitos, vias de sinalização celular disparadas pela exposição ao choque hiperosmótico e estresse oxidativo foram investigadas em células HaCaT tratadas com sorbitol e peróxido de hidrogênio (H2O2) respectivamente. Os resultados obtidos neste estudo demonstraram que, em ambos os modelos, a redução da viabilidade celular dependeu da dose e do tempo de exposição ao agente extressor, apresentando valores de IC50 de aproximadamente 1 mol/L de sorbitol e 2 mmol/L de H2O2 após 2 e 4 h de exposição respectivamente. Os danos causados foram irreversíveis e estão associados à ativação da via intrínseca de morte celular apoptótica, acompanhada de perda da integridade da membrana lisossomal, extravasamento de catepsina B para o citosol e alterações morfológicas atípicas no citoesqueleto, principalmente no arranjo dos filamentos de actina. A investigação do status de funcionamento de proteínas quinases ativadas por mitógenos (MAPKs) e do estado redox celular indicou que esses eventos foram mediados por espécies reativas de oxigênio e pela ação da quinase c-Jun N-terminal (JNK). Adicionalmente, a exposição dos queratinócitos aos diferentes estímulos estressores foi acompanhada de ativação da proteína tirosina fosfatase de baixa massa molecular (LMWPTP), cuja relevância nos estudos de biologia celular aumentou nos últimos anos. A LMWPTP atua em importantes vias de sinalização que estão associadas à sobrevivência e morte celular. Cientificamente, este estudo é pioneiro ao demonstrar alterações no citoesqueleto e ação de proteínas quinases e fosfatases nos mecanismos que determinam o destino de queratinócitos expostos ao choque hiperosmótico e ao estresse oxidativo. De fato, o melhor conhecimento da relação entre as vias de sobrevivência e morte celular em queratinócitos é fundamental para promover o desenvolvimento de novas estratégias terapêuticas aplicadas às doenças dermatológicas. Desta maneira, o presente trabalho apresenta resultados inéditos, contribuindo no conhecimento da biologia dos queratinócitos e com sua aplicação no desenvolvimento da terapia dermatológica / Abstract: The epidermis is constantly confronted with multiple environmental stressors. Changes in humidity or exposition to UV radiation affect the redox state and osmotic balance, modifying the physiological characteristics of the skin. In response to different stresses, epidermal keratinocytes can activate distinct signaling pathways and the balance between death and life signals will determine the cell fate, leading to programmed cell death or cell survival. In order to determine the possible molecular targets associated to death and survival of keratinocytes, the signaling pathways activated by the exposition of HaCaT cells to sorbitol (hyperosmotic shock) and H2O2 (oxidative stress) were investigated. The results showed that in both models the reduction in cellular viability was time and dose-dependent, displaying IC50 values of 1 mol/L for sorbitol and 2 mmol/L for H2O2 after 2 and 4 h of exposition to the stressors, respectively. The damages caused by the stressors were irreversible and associated to the induction of the intrinsic apoptotic pathway, accompanied by the loss of lisosomal membrane integrity, release of cathepsin B to cytosol and atypical morphological alterations in cytoskeleton, particularly in the arrangement of actin filaments. Analysis of the functional status of mitogen-activated protein kinases (MAPKs) and the cellular redox state showed that such events were mediated by reactive oxygen species and occurred through c-Jun N-terminal kinase (JNK) activation. Additionally, exposure of keratinocytes to the different stress inducers was followed by low molecular weight tyrosine protein phosphatase (LMWPTP) activation, which is responsible for the regulation of important signaling pathways associated to cell survival and death. It is important to highlight the novelty of these results showing alterations in the cytoskeleton and the action of protein kinases and phosphatases during exposure of keratinocytes to hyperosmotic and oxidative stresses. In fact, the development of more efficacious therapies against skin diseases depends on the establishment of the relationships between the survival and death signaling pathways in keratinocytes. In this direction, this work contributes to a better understanding of the keratinocyte biology and the improvement of traditional dermatological therapies / Doutorado / Bioquimica / Doutor em Biologia Funcional e Molecular

Orientador: Jörg Kobarg / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-27T05:36:59Z (GMT). No. of bitstreams: 1 MeloHanchuk_TalitaDiniz_D.pdf: 15551209 bytes, checksum: 4fffad9810f85e106f425f0bfbde833b (MD5) Previous issue date: 2015 / Resumo: Membros da família das Neks são cruciais para o início da mitose em eucariotos. Têm sido funcionalmente atribuídas a todas as 11 Neks humanas uma das três principais funções estabelecidas para esta família em mamíferos: (1) centríolos / divisão celular; (2) funções no cílio primário / ciliopatias; e (3) resposta à danos no DNA (DDR). No artigo de revisão (artigo I), relatamos uma análise detalhada atual sobre cada uma das 11 Neks. A hipótese é que as Neks possam conectar elementos reguladores que permitem o refinamento e a sincronização de eventos celulares. Dentre os membros desta família, Nek5 é a cinase mais negligenciada. Ensaios de duplo híbrido em leveduras (Y2H) foram realizados para identificar e caracterizar parceiros de interação Nek5; e proteínas mitocondriais foram observadas (artigo II). Ensaios de apoptose mostraram efeitos protetores na morte celular após tratamento com tapsigargina (2 ?M) de células HEK293T que superexpressam a hNek5, bem como a diminuição na formação de Espécies Reativas de Oxigênio após 4 horas de tratamento. A atividade da cadeia respiratória mitocondrial estava diminuída após superexpressão de hNek5, especialmente nas etapas de transferência de elétrons do TMPD para o citocromo c e no complexo II. O Y2H permitiu também a identificação da poliglutamilase de proteínas TTLL4 como um parceiro de Nek5 (artigo III). Células silenciadas para a Nek5, assim como células que expressam a versão "kinase dead" de Nek5, apresentaram por western blot e ensaio in vitro de atividade poliglutamilação um aumento na poliglutamilação de proteínas após transfecção com TTLL4. Em conclusão, nossos dados sugerem pela primeira vez a localização mitocondrial e a participação de Nek5 na morte celular e no processo poliglutamilação diminuindo a atividade de TTLL4 através de sua fosforilação inibitória / Abstract: Members of the Nek Family are crucial for the initiation of mitosis eukaryotes. All 11 human Neks have been functionally assigned to one of the three core functions established for this family in mammals: (1) centrioles/mitosis; (2) primary ciliary function/ciliopathies; and (3) DNA damage response (DDR). In the core section of the review (article I), we report the current detailed functional knowledge on each of the 11 Neks. We raise the hypothesis that Neks may be the connecting regulatory elements that allow the cell to fine tune and synchronize cellular events. Nek5 is the most neglected among members of the Nek kinases family. A yeast two-hybrid (Y2H) screen was performed to identify and characterize Nek5 interaction partners and mitochondrial proteins were retrieved (article 2). Apoptosis assay showed protective effects of hNek5 over-expression from Hek293-T¿s cell death after thapsigargin treatment (2 ?M) as well as an increase in ROS formation after 4 hours of treatment. Mitochondrial respiratory chain activity was found decreased upon hNek5 over-expression especially at the electrons transfer steps from TMPD to cytochrome c and at the complex II. The yeast two-hybrid allowed also the identification o TTLL4 as a Nek5 partner (article 3). Nek5 silenced cells as well as cells expressing a "kinase dead" version of Nek5, displayed an increase in polyglutamylation of proteins after TTLL4 transfection by western blot and in vitro polyglutamylation activity assay. In conclusion, our data suggest for the first time mitochondrial localization and functions for Nek5 and its participation in cell death and cell respiration regulation. This work also showed the function of Nek5 in the polyglutamylation process decreasing the role of TTLL4 through inhibitory phosphorylation by Nek5 / Doutorado / Bioquimica / Doutora em Biologia Funcional e Molecular

Peptídeos intracelulares são constantemente produzidos pelo sistema ubiquitina proteassomo e muitos são provavelmente funcionais. Aqui, um nonapeptídio derivado da ciclina-D2, específica da transição G1/S, chamado \"pep5\" mostrou um aumento específico durante a fase S do ciclo celular em células HeLa. O pep5 (50-100 μM) induziu a morte celular em células HeLa e em várias outras células tumorais, mas isso só ocorreu quando o pep5 foi sintetizado acoplado ao peptídeo penetrador de células (pep5-cpp). In vivo, o pep5-cpp reduziu o volume do glioblastoma C6 de ratos Wistar em cerca de 50%. A acetilação reduziu a potência do pep5-cpp, enquanto substituições Leu-Ala aboliram totalmente a atividade deste peptídeo. Os resultados de caracterização inicial do mecanismo de morte celular indizida pelo pep5 incluem ativação de caspases 3/7 e 9, inibição da fosforilação Akt2 e inibição da atividade do proteassomo. Esses dados colaboram com a hipótese da função de peptídeos intracelulares na sinalização. / Intracellular peptides are constantly produced by the ubiquitinproteasome system and many are probably functional. Here, a nonapeptide of G1/S-specific cyclin-D2 named pep5 showed a specific increase during the S phase of HeLa cell cycle. Pep5 (50-100 μM) induced cell death in HeLa and in several other tumor cells, only when it was fused to a cell penetrating peptide (pep5-cpp). In vivo, the pep5-cpp reduced the volume of the rat C6 glioblastoma by almost 50%. Acetylation reduced the potency of the pep5-cpp while Leu-Ala substitutions totally abolished the pep5 activity. Findings from the initial characterization of the cell death mechanism of pep5 include caspase 3/7 and 9 activation, inhibition of Akt2 phosphorylation and inhibition of proteasome activity. These data further support the intracellular function of peptides.

Une des caractéristiques des cellules tumorales est leur habileté à échapper à la mort cellulaire. Pour y parvenir, elles ont développé une stratégie consistant à éliminer sélectivement les mitochondries endommagées par un processus de mitophagie. L’acteur principal de la mitophagie est l’ubiquitine ligase Parkin ; mais elle est mutée ou absente dans la majorité des cancers. Nous avons découvert qu’une autre ligase, ARIH1, appartenant à la même famille des RBR ligases que Parkin, est capable d’induire la mitophagie en réponse à un stress. Contrairement à Parkin, ARIH1 est surexprimée dans de nombreux cancers, notamment dans les cancers du poumon permettant ainsi une augmentation de la mitophagie conférant ainsi à ces cellules une résistance au stress induit par des agents chimiothérapeutiques. La mort cellulaire la mieux caractérisée est l’apoptose qui est directement liée à l’activation de caspases. Il a pourtant été établi qu’une inhibition des caspases ne permet pas d’empêcher la mort cellulaire car il existe la « mort cellulaire indépendante des caspases » ou CICD. Cependant, sa définition moléculaire précise reste toujours inconnue. Ainsi dans ce but, un criblage siRNA pan génomique a révélé l’importance de la voie ubiquitine/protéasome. Nous avons pu identifier en particulier une enzyme E3 ligase comme étant protectrice de la CICD. Cette enzyme est surexprimée dans de nombreux cancers et pourrait permettre aux cellules cancéreuses de résister à la CICD et favoriser la progression tumorale. En résumé, ce travail a permis de souligner l’importance des ubiquitines ligases dans les mécanismes d’échappement à la mort cellulaire mis en place par les cellules cancéreuses. / One of the hallmarks of tumor cells is their ability to escape cell death.To achieve this, they have developed a strategy of selectively removing damaged mitochondria by a process of mitophagy. The main actor of mitophagy is the ubiquitin ligase Parkin; but it is mutated or absent in the majority of cancers. We have discovered that another ligase, ARIH1, belonging to the same family of RBR ligases as Parkin, is capable of inducing mitophagy in response to stress. In contrast to Parkin, ARIH1 is overexpressed in many cancers, especially in lung cancer, allowing an increase in mitophagy conferring resistance to stress induced by chemotherapeutic agents. The most characterized cell death pathway is apoptosis, which is directly related to caspases activation. However, it has been established, that caspase inhibition does not prevent cell death because there is another type of cell death called "caspase-independent cell death" or CICD. However, its precise molecular definition is still unknown. Thus for this purpose, pan-genomic siRNA screening was performed and revealed the importance of the ubiquitin / proteasome pathway. In particular, we have been able to identify an enzyme E3 ligase as being protective towards CICD. This enzyme is overexpressed in many cancers and could allow cancer cells to resist CICD and promote tumor progression. In summary, this work has highlighted the importance of ubiquitin ligases in the escape mechanisms to cell death implemented by cancer cells.

Mes travaux de recherche révèlent une nouvelle voie de signalisation qui est impliquée dans l'étape d'internalisation cellulaire. Cette voie de signalisation cellulaire nécessite une libération d'ATP ainsi que l'activation de récepteur purinergique au niveau des cellules cannibales et va aboutir à l'élimination de la cellule internalisée. Nous avons également défini au cours de ces travaux le devenir de la cellule cannibale et préciser au cours d'expérimentations in vivo l'activité oncosuppressive du cannibalisme cellulaire. Nous avons également observé ce processus au niveau de biopsies tumorales obtenues à partir de patients ayant un cancer du sein et ayant reçu un traitement néo-adjuvant et avons révélé que sa détection pouvait prédire l’efficacité d'un traitement néo-adjuvant. L’ensemble de ces résultats révèle les bases moléculaires du cannibalisme cellulaire et précise le rôle du cannibalisme cellulaire lors du développement tumoral. / My research reveals a new signaling pathway which is involved in the cellular internalization. This signaling pathway requires ATP release and purinergic receptor activation at the level of cannibal cells and will lead to the elimination of internalized cell. We also defined during this work the future of the cannibal cell and specify during experiments in vivo the tumor suppressor activity of cellular cannibalism. We also observed this process in tumor biopsies obtained from patients with breast cancer who received neoadjuvant treatment and have revealed that its detection could predict the efficacy of neoadjuvant treatment. Theses results reveal molecular bases of cellular cannibalism and indicate the role of cellular cannibalism during tumor development.

After dendrite transection, two primary injury current pathways may acount for cell death: (1) the lesion current at the site of injury and (2) the voltage sensitive calcium channels along the dendrite. Lesions were made with a laser microbeam in mouse spinal monolayer cell cultures. Polylysine was tried as a positively charged "molecular bandage" to block the lesion current. The calcium channel blockers, verapamil and nifedipine, were used to reduce the calcium channel current. Control toxicity curves were obtained for all three compounds. The results show that neither verapamil, nifedipine, nor polylysine (MW: 3,300) protect nerve cells after dendrite amputation 100 ptm from the soma. The data also indicate that these compounds do not slow the process of cell death after such physical trauma.

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第21977号 / 生博第417号 / 新制||生||55(附属図書館) / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 垣塚 彰, 教授 HEJNA James, 教授 豊島 文子 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22329号 / 医博第4570号 / 新制||医||1041(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 坂井 義治, 教授 斎藤 通紀, 教授 岩田 想 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Necroptosis, a type of regulated necrotic cell death, involves cell membrane permeabilization and has been implicated in various acute and chronic pro-inflammatory diseases, including ischemia-reperfusion injury and neurodegenerative diseases. By using in vitro reconstitution studies and a chemical inhibitor, the kinase activity of the serine/threonine kinase RIPK1 had been shown to regulate necroptotic signaling downstream of TNF and Toll-like receptors (TLRs). To investigate the contribution of RIPK1 kinase activity to inflammation and necroptosis in vivo, we generated kinase inactive RIPK1 knock-in mice. Utilizing fibroblasts and macrophages from these mice, we demonstrate that RIPK1 kinase activity is required for necroptotic complex formation and death induction downstream of TNFR1 and TLRs 3 and 4. We show that RIPK1 kinase inactive mice are resistant to TNF-induced shock and exhibit impaired upregulation of TNF-induced cytokines and chemokines in vitro and in vivo. By using bone marrow reconstitution experiments, we demonstrate that RIPK1 kinase activity in a non-hematopoietic lineage drives TNF-induced lethality. We establish that RIPK1 kinase activity is required for TNF-induced increases in intestinal and vascular permeability and clotting, and implicate endothelial cell necroptosis as an underlying factor contributing to TNF/zVAD-induced shock. Thus, work in this thesis reveals that RIPK1 kinase inhibitors may have promise in treating shock and sepsis.

Glioblastoma is the most common primary brain tumor in humans and has a poor prognosis. Current therapies are not curative. Oncolytic viruses (OVs) are being investigated as tools to induce immunogenic cell death (ICD), cell death capable of activating the immune system. Semliki Forest Virus (SFV) strain 4 is an OV being investigated to treat glioblastoma. Previous studies in our lab have shown that SFV4 can induce ICD in human osteosarcoma (HOS) cells and ongoing in vivo studies show that SFV4 infected GL261 cell vaccination providesprotective immunity in mouse models. This study aimed to characterize the ICD induced by SFV4in glioblastoma cell lines, namely GL261, SB28 and CT2A, and to explain some of our in vivoobservations, namely why vaccination with SFV infected GL261 provides protective immunity but vaccination with infected CT2A and SB28 does not. Our in vitro studies found that GL261 is resistant to SFV4 while SB28 and CT2A are susceptible. We show that the virus can replicate in all three cell lines as seen by the presence of dsRNA, but that viral translation is delayed or inhibited in GL261 cells as not all cells positive for dsRNA were positive for SFV4 protein. Additionally, the type I interferon (IFN) pathway, responsible for antiviral defense, was highly upregulated in CT2A and SB28 but not as much in GL261 after infectionas seen by surveying IFIT1, IFITM3 and IFN-beta genes. Interferon stimulated genes (ISG) like CXCL10, a chemoattractant, was highly upregulated in GL261 after infection and might account for the protective immunity seen in vivo after vaccination. PDL1, an interferon stimulated gene responsible for self-tolerance, was highly upregulated in CT2A after infection. The IFN-beta ELISA revealed that both infected and uninfected GL261 cells produce IFN-beta suggesting a constitutively active pathway. Our DC phagocytosis assay showed that SFV4 infection of CT2A and SB28 cells induced a significant increase in DC phagocytosis and SFV4 infection of all three cell lines significantly increased DC maturation. We conclude that SFV4 infection of GL261 cells may induce ICD in vivo through a persistent viral infection and increased expression of CXCL10.